Fig. 6.
The protein phosphatase activity of CinF is important for its role in C. burnetii virulence. (A) KD of cinF in C. burnetii and complementation with a codon-changed allele. The level of CinF was probed in bacteria grown in a bacteriological medium. An ∼80-kDa protein nonspecifically recognized by the CinF antibodies was used as a loading control (Upper). The relative abundance of CinF was determined by the intensity ratio between the target band and the nonspecific band (Lower). Results shown were from three independent experiments. (B) KD of cinF affects intracellular replication of C. burnetii. The indicated bacterial strains were used to infect HeLa cells at a multiplicity of infection (MOI) of 100, and the growth of the bacteria was determined by measuring genome equivalents. Note that KD of cinF led to less replication, a defect that can be restored by expressing a wild-type allele of cinF. (C and D) Kinetics of IκBα, p-IκBα, and CinF translocation at the third day (C) and fourth day (D) after bacterial uptake. THP-1 cells infected with the indicated C. burnetii strains were probed for p-IκBα, IκBα, and translocated CinF. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as a loading control. The insoluble fractions were used to probe for CinF associated with the bacteria with the metabolic enzyme isocitrate dehydrogenase (ICDH) as a loading control. (E and F) The translocation of p65 in cells infected with relevant C. burnetii strains. THP-1 cells infected with the indicated strains as described in C were stained for p65, and nuclear localization of the protein was scored under a fluorescence microscope (refer to SI Appendix, Fig. S5A for representative images) (E). Results shown were from three independent experiments done in triplicate. Statistical analysis was performed by Student’s t test. Similarly infected cells were subjected to fractionation to obtain the nuclear fractions, which were probed for C. burnetii and p65, respectively (F). GAPDH and histone H3 were detected as controls. Results shown were one representative from three independent experiments with similar results. (G) Knocking out p65 allows the cinF KD strain to replicate intracellularly at rates similar to the wild-type strain. The wild-type and cinF KD strains were used to infect HeLa cells (solid lines) or the p65−/− line (dashed lines) at an MOI of 100. The growth of the bacteria was monitored by measuring genome equivalents. Note that the KD strain grew at rates similar to those of the wild-type strain in the p65−/− cell line. Data shown were one representative from three independent experiments with similar results. (H) Expression of CinF in L. pneumophila led to inhibition of NF-κB activation induced by bacterial infection. The indicated L. pneumophila strains were used to infected HEK293 cells transfected with the luciferase reporter plasmids. The induction was measured 6 h after infection (Upper). The level of IκBα in infected cells was probed by immunoblotting. CinF or CinFY362A translocated into infected cells was also probed, and GAPDH was detected as a loading control (Lower). When applicable, statistical analysis was performed by Student’s t test.